Collagen which constitutes connective tissues of animals is composed of three polypeptide chains, each basic unit of which has a molecular weight of about 95,000. These polypeptide chains form a counterclockwise triplex spiral structure. The amino acid sequence of each polypeptide chain of the collagen molecule is a repetition of Gly-Pro-X-Gly (wherein the three-letter code representing amino acid residues SEQ ID No: 1) used herein are those according to IUPAC-IUB standards and X represents various amino acid residues) and the polypeptides are intramolecularly or intermolecularly cross-linked. This specific spiral structure of collagen brings about tough mechanical properties and chemical stability and, therefore, collagen resists degradation by ordinary proteases and only a collagenase can degrade collagen.
A collagenase does not act on ordinary proteins, but acts on only the above collagen or its modified product, gelatin. Collagenases are produced by microorganisms and, among these collagenases, the study on the collagenase known as Achromobacter collagenase which is derived from Vibrio alginolyticus chemovar. iophagus is most advanced. It has been known that this collagenase has a higher specific activity in comparison with collagenases derived from other sources [V. Keil-Dlouha and B. Keil, Biochim. Biophys. Acta, 522, 218-228 (1978)]. Achromobacter collagenase has a molecular weight of 110,000 and is stable at pH 6 to 7. The optimum pH is about pH 7.4. The collagenase, which is inactivated by EDTA and o-phenanthroline [V. Keil-Dlouha, Biochim. Biophys. Acta, 429, 239-251 (1976)], is a metalloprotease containing zinc, and breaks the synthetic substrate, PZ-Pro-Leu-Gly-Pro-D-Arg, between Leu and Gly [B. Keil, A. M. Gilles, A. Lecroisey, N. Hurion and N. T. tong, FEBS Lett., 56, 292-296 (1975); A. Lecroisey, V. Keil-Dlouha, D. R. Woods, D. Perrin and B. Keil, FEBS Lett., 59, 167-172 (1975); N. T. Tong, A. Tsugita and V. Keil-Dlouha, Biochim. Biophys. Acta, 874, 296-304 (1986)].
In view of the specific property of a collagenase, various uses have been expected and realized. For example, a collagenase is used for treatment of various injuries of any substrate having a structure rich in collagen. Examples of such injuries include burn, ulcer, scab, white hard scab of collagen base, cheloid, necrosis, particularly, necrosis by decubitus or ulcer, and the like.
A collagenase is also used for treatment of dental caries. Namely, the dental pulp is mainly composed of a dense calcareous material and collagen. In the case of dental caries, a tooth is cracked or a hole is made and calcium is leaked therefrom. Accordingly, the calcareous material is lost, and the remaining frame becomes porous and is liable to be a hotbed of bacterial infection. However, since a collagenase dissolves the porous collagen, the hotbed can be removed by washing with water. A collagenase does not act on healthy calcareous collagen.
In addition, a collagenase can be used as an agent for making meat tender. Toughness of meat is mainly caused by tendon, the main component of which is collagen. Proteases such as papain and the like are used to make meat tender by degrading tendon. However, collagen is hardly degraded by ordinary proteases. On the other hand, non-specific proteases such as papain also degrade proteins such as actin, myosin and the like which have great influence on the texture 0f meat. Therefore, the texture of meat is destroyed by treatment with papain. In this respect, since a collagenase degrades only collagen which causes toughness of meat, but does not degrade other proteins which have great influence on the texture of meat, the enzyme is a protease most suitable for an agent for making meat tender.
In the use of a collagenase for the above purposes, there is a problem that it is very difficult to obtain a collagenase at a low cost. Namely, in order to obtain Achromobacter collagenase, its producer, Vibrio alginolyticus, is cultivated and the collagenase is recovered from the culture solution and purified. However, in this respect, there is a problem that the yield of collagenase by the producer is very low such as 10 mg/liter. Further, there is another problem that any collagenase is not produced by the producer unless a certain specific inducing substance is added to a culture medium. Thus, it is very difficult to obtain the collagenase in a large amount at a low cost.
Although it is possible to employ genetic engineering techniques to solve these problems, no gene of Achromobacter collagenase is yet available. Therefore, no genetic engineering technique can be employed to produce the enzyme in a large amount.